Angiotensin-converting enzyme inhibitory peptides

ABSTRACT

It is intended to provide ACE inhibitory tripeptides which are not easily digested by digestive enzymes after being orally taken and thus have fewer tendencies to lose their ACE inhibitory activity in vivo. 
     More specifically, 3 tripeptides having an ACE inhibitory activity and showing a hypotensive effect in an animal experiment are discovered from a thermolysin digestion product of sesame. These tripeptides respectively have amino acid sequences Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyr and show an angiotensin converting enzyme inhibitory activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/549,176 filed Aug. 14, 2006, which is a national stage ofInternational PCT Application No. PCT/JP2004/003588, filed Mar. 17,2004, which claims benefit of Japanese Patent Application No.2003-74488, filed Mar. 18, 2003.

FIELD OF THE INVENTION

This invention relates to peptides which inhibit angiotensin convertingenzyme and which are therefore useful as ingredients of health foods,drugs, etc. having a hypotensive effect.

PRIOR ART

The number of patients with hypertension, which is a typical example oflife-style related diseases, is increasing year by year. It is knownthat hypertension induces various complications such as cerebralhemorrhage, subarachnoid hemorrhage, cerebral infarction, myocardialinfarction, angina, nephrosclerosis and so on. Thus, various studieshave been made on the onset mechanism of hypertension.

As blood pressure-regulatory systems, the renin-angiotensin systemrelating to the elevation of blood pressure and the kallikrein-kininsystem relating to the reduction of blood pressure play important roles.In the renin-angiotensin system, angiotensinogen secreted from the liveris converted into angiotensin I by renin produced in the kidney.Angiotensin I is further converted into angiotensin II by angiotensinconverting enzyme (ACE). Angiotensin II induces contraction of smoothvascular muscles and thus elevates blood pressure. On the other hand,kallikrein in the hypotensive system acts on kininogen and thus producesbradykinin. Bradykinin has a vasodilating effect and lowers bloodpressure. However, ACE has an effect of degrading bradykinin. That is tosay, it is known that ACE participates in the elevation of bloodpressure through the above-described two effects, i.e., producingangiotensin II which is a vasopressor peptide and inactivatingbradykinin which is a hypotensive peptide. Therefore, it will bepossible to reduce elevation of blood pressure by suppressing the enzymeactivity of ACE. Proline derivatives such as captopril and enalaprildeveloped as ACE inhibitors have been widely employed in treatinghypertension.

In recent years, it has been reported that peptides obtained bydigesting food materials with enzymes have an ACE inhibitory activity.For example, there have been reported a large number of such digestionproducts, e.g., a collagenase digestion product of gelatin (JapanesePatent Public Disclosure SHO 52-148631), a trypsin digestion product ofcasein (Japanese Patent Public Disclosure SHO 58-109425, Japanese PatentPublic Disclosure SHO 59-44323, Japanese Patent Public Disclosure SHO60-23086, Japanese Patent Public Disclosure SHO 60-23087, JapanesePatent Public Disclosure SHO 61-36226 and Japanese Patent PublicDisclosure SHO 61-36227), a thermolysin digestion product of γ-zein(Japanese Patent Public Disclosure SHO 2-32127), a pepsin digestionproduct of sardine muscle (Japanese Patent Public Disclosure HEI3-11097), a thermolysin digestion product of dried bonito (JapanesePatent Public Disclosure HEI 4-144696), a thermolysin digestion productof sesame protein (Japanese Patent Public Disclosure HEI 8-231588), apepsin digestion product of κ-casein (Japanese Patent Public Disclosure8-269088) and so on.

These ACE inhibitory peptides, being of food origin, have significantadvantages, i.e., they pose few problems of safety (i.e., side effects,toxicity, etc.) and are edible like common foods. However, it has beenreported that the above-described peptide products mainly comprisepeptides of 5 or more amino acids (Japanese Patent Public Disclosure SHO52-148631, Japanese Patent Public Disclosure SHO 58-109425, JapanesePatent Public Disclosure SHO 59-44323, Japanese Patent Publication SHO60-23086, Japanese Patent Public Disclosure SHO 61-36226, JapanesePatent Public Disclosure SHO 61-36227, Japanese Patent Public DisclosureHEI 3-11097, Japanese Patent No. 3135812 and Japanese Patent PublicDisclosure HEI 8-269088). It has been pointed out that peptidesconsisted of longer amino acid chains cannot achieve a hypotensiveeffect of the level expected based on the strong ACE inhibitory activityin vitro, probably because they are susceptible to digestion bydigestive enzymes in the body and thus lose the ACE inhibitory activity,or, even if they remain not digested, they are not easily absorbedbecause of their bulky molecular structures.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide ACEinhibitory tripeptides which are not easily digested by digestiveenzymes when taken orally and thus have fewer tendencies to lose theirACE inhibitory activity in vivo.

In the present invention, it is also intended to provide edible(food/drink) compositions, angiotensin converting enzyme inhibitors andhypotensive agents comprising one or more of the above-describedtripeptides.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the results of an examination on thehypotensive effects of the peptides according to the present inventionwith the use of spontaneous hypotensive rats.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors searched for ACE inhibitory peptides consisted ofnot more than 3 amino acids, assuming that thermolysin digestionproducts of food materials would contain peptides capable of overcomingthe above-described problems. As a result, they succeeded in discovering3 tripeptides in a thermolysin digestion product of sesame, saidtripeptides having an ACE inhibitory activity and showing a hypotensiveeffect in an animal experiment. The present invention was accomplishedbased on these findings.

Accordingly, the present invention provides tripeptides respectivelyhaving the amino acid sequences Leu-Ser-Ala, Val-Ile-Tyr and Leu-Val-Tyrand showing an angiotensin converting enzyme inhibitory activity.

The present invention further provides edible compositions, angiotensinconverting enzyme inhibitors and hypotensive agents containing one ormore of the above-described tripeptides.

The tripeptides according to the present invention may be produced bychemical synthesis. However, in an embodiment of the invention whereinthe tripeptides are added to foods, drinks or oral drugs to exploittheir ACE inhibitory activity, it is preferred to produce an ediblecomposition enriched with at least one of the above-described 3tripeptides by digesting vegetable protein originating in sesame or thelike with thermolysin and further purifying the same.

As a vegetable protein source, use can be made of protein-rich planttissues (preferably seeds), for example, cereals such as rice, wheat,barely, oat and corn, or beans such as kidney bean, broad bean, soybeanand mung bean and sesame.

When the peptides according to the invention are to be obtained bydigestion with thermolysin, the treatment procedure varies depending onthe properties of the starting material. It is preferred that, as apretreatment, the material is first defatted by, for example, removingthe juice by squeezing or extracting the fat with a solvent such as analcohol, acetone, hexane, etc. To improve the efficiency of thedigestion of the starting material with thermolysin, it is alsopreferred that the starting material be finely milled and then suspendedin water under stirring. In the case of a hardly soluble protein, it isalso possible to employ another pretreatment such as addition of sodiumhydroxide or heating to thereby uniformly dissolve or suspend theprotein. Then thermolysin is added thereto in an appropriate amount,preferably from 500 to 50000 PU per g of the protein and the proteindigestion reaction is carried out at pH 5 to 9, at a temperature of 10to 80° C., for 0.5 to 48 hours either in a stationary state or understirring. (“PU” means “protease unit” and 1 PU is defined as the amountof an enzyme giving an increase in non-protein Folin's color equivalentto 1 μg of tyrosine per min using milk casein as the substrate at pH 7.2and at 35° C.) To examine whether or not sufficient progress of thereaction has been made (i.e., the reaction is sufficient for obtainingthe purposed tripeptides), use can be made of a method comprising“applying the liquid reaction mixture to high-performance liquidchromatography using an ODS column and determining the elution patternby measuring the absorbance at 210 nm”. The reaction is ceased byadding, for example, hydrochloric acid. Alternatively, the thermolysinmay be inactivated by heating. It is also possible to cease the reactionby both the adding hydrochloric acid and heating. The liquid reactionmixture is subjected to centrifugation, filtration, etc. and theprecipitate is removed. The filtrate thus obtained is neutralized withsodium hydroxide or hydrochloric acid and then concentrated. Further,off-flavor (for example, bitterness, harshness, offensive odor, etc.)can be removed if necessary by treating it with activated charcoal. Thesesame peptides thus obtained contain Leu-Ser-Ala, Val-Ile-Tyr andLeu-Val-Tyr each in an amount of from 0.001% by weight to 0.1% byweight.

The thermolysin digestion product obtained in the above-described mannercan be used as the tripeptide composition of the invention with orwithout a further treatment with an ion exchange resin, a high-porouspolymer resin, etc. to remove high-molecular weight proteins, to therebyprovide a partially purified product rich in the tripeptides of thepresent invention. These digestion product and partially purifiedproduct in general will be sometimes referred to hereinafter as“tripeptide-rich composition”. Such a composition may be furthertreated, if necessary, by activated charcoal to remove off-flavor (forexample, bitterness, harshness, offensive smell, etc.) before using.

To obtain a purified preparation of the peptides of the invention, theabove-described concentrate is subjected to gel filtration columnchromatography, chromatography with the use of an ion exchange resin ora high-porous polymer resin, affinity chromatography, etc. and peptidefractions of the invention having the ACE inhibitory activity arecombined. Next, the combined active fractions can be purified by amethod commonly employed in purifying peptides, for example,high-performance liquid chromatography with the use of a reversed phasecolumn such as an ODS column or a C30 column to thereby provide singleforms of the peptides in a substantially pure state. The tripeptides ofthe invention can be obtained not only from sesame (e.g., Sesamumindicum L.) but also from cereals such as rice (e.g., Oryza sativa L.),wheat (e.g., Triticum aestivum L., T. durum Desf., T. turgidum L., T.pyramidale (Delile) Perciv. non Delile ex Schult, T. abyssinicumVavilov, and T. carthlicum Nevski), barley (e.g., Hordeum vulgare L.),oat (e.g., Avena sativa L.) or corn (e.g., Zea mays L.), or beans suchas kidney bean (e.g., Phaseolus vulgaris L.), broad bean (e.g., Viciafaba L.), soybean (e.g., Glycine max (L.) Merrill) or mung bean (e.g.,Vigna radiata (L.) R. Wilcz.) by the method as described above. The ACEinhibitory activity of the tripeptides or tripeptide-rich compositionscan be measured by, for example, an in vitro test method and/or an invivo test method as will be described in Examples hereinafter.

When each peptide of the invention is prepared by chemical synthesis,the synthesis can be carried out by any of the solid phase method andthe liquid phase method conventionally employed in synthesizing apeptide. The peptide of the invention obtained by the synthesis can bepurified by a purification procedure commonly employed, for example,reverse phase high-performance liquid chromatography, chromatographywith the use of an ion exchange resin or a high-porous polymer resin,affinity chromatography, etc.

The tripeptides thus obtained and the composition enriched with thetripeptides have a strong activity of inhibiting ACE and exhibit thestrong ACE inhibitory effect when they are taken orally. Therefore, theyare useful as highly potent ACE inhibitors. Moreover, they are easilyabsorbed via the gastrointestinal tract and are relatively stable underheat. Due to these characteristics, they are also applicable to foods,drinks and medicinal preparations in various forms.

Accordingly, the present invention provides an edible composition whichcomprises one or more of the above-described tripeptides and which isexpected to have an angiotensin converting enzyme inhibitory effect, anangiotensin converting enzyme inhibitor and a hypotensive agentcontaining one or more of the above-described tripeptides.

In a case where one or more of the tripeptides of the invention are usedin foods, drinks, drugs, etc., use may be made of a tripeptidesufficiently purified from the thermolysin digestion product of theprotein fraction of sesame, etc., or use may be made of a chemicallysynthesized product. Alternatively, since the tripeptides of theinvention have a high stability and a strong ACE inhibitory activity,the above-described partially purified product or the thermolysindigestion product or the partially purified product thereof may be usedas such as a tripeptide-rich composition; in such a case also, asufficient ACE inhibitory activity will be obtained, and hence, this isa preferred embodiment of the invention.

The edible composition according to the present invention is produced byadding one or more of the above-described tripeptides in an amount offrom 0.001 mg to 100 mg, preferably from 0.01 mg to 20 mg and stillpreferably from 0.1 mg to 10 mg in a single intake dose. The tripeptidesof the invention are in the form of a solid or a powder which can beeasily handled and are highly soluble in water. Also, the tripeptidescan be well absorbed via the gastrointestinal tract. Therefore, thetripeptides may be added to foods at any stage by any method withoutparticular restriction. That is to say, the tripeptides can be added inthe form of a powder, a solution, a suspension, etc. at the startingstage, the intermediate stage or the final stage of a food productionprocess by using a method commonly employed in the field of foodindustry. Temporary, intermittent, continuous or daily intake of theedible composition containing the tripeptides of the invention makes itpossible to inhibit angiotensin converting enzyme and obtain, forexample, a hypotensive effect. The foods and drinks may be in the formof, for example, a solid, a semifluid or a fluid. Examples of solidfoods include general foods and health foods in the forms of biscuits,sheets, pills such as tablets and capsules, granules, powders and so on.Examples of semifluid foods include products in the forms of pastes,jellies, gels and so on. Examples of fluid foods include general drinkproducts and health drinks in the forms of juices, cold drinks, teadrinks, tonic drinks and so on. Such foods or drinks may be supplied inthe form of a nutrition supplemental drink or a seasoning to enable usto continuously take the tripeptides of the invention, therebysuppressing the risk of blood pressure elevation.

The medicinal composition of the invention contains the tripeptides ofthe invention in an amount similar to the edible composition asdescribed above. The medicinal composition of the invention can betemporarily administered to a hypertensive patient to suppress theangiotensin converting enzyme in the body and thereby to obtain ahypotensive effect. Alternatively, the medicinal composition of theinvention can be continuously administered safely, since the activeingredient originates in a natural material. As an example of thediseases which can be treated and/or prevented by the medicinalcomposition of the invention, hypertension can be mentioned. It ispreferable that the medicinal composition is in the form of an oralpreparation such as tablets, capsules, dusts, granules or syrups.Examples of preparations for parenteral administration include asepticsolutions to be administered intravenously, intraarterially,subcutaneously, intramuscularly or intranasally. Such a solution may bein the form of a dry solid which is to be dissolved before using. Aninjection preparation can be produced by dissolving an effective amountof the tripeptide in physiological saline and treating under asepticconditions as commonly employed in producing injection preparations.

EXAMPLES

Now, the present invention will be described in greater detail byreference to the following Examples.

Method of Measuring Ace Inhibitory Activity

In the present invention, the ACE inhibitory activity (IC₅₀) wasmeasured in accordance with the following method.

-   -   Buffer: 0.1 M HEPES, 0.3 M NaCl, 0.01% Triton-X (pH 8.3).    -   Enzyme: ACE from rabbit lung (Sigma).        -   Dissolved in the above buffer and adjusted to a            concentration of 1 mU/50 μl.    -   Substrate: Bz-Gly-His-Leu H₂O (Peptide Institute Inc.).        -   8.95 mg of the substrate was dissolved in 1 ml of dimethyl            sulfoxide and further diluted 5-fold with water (final            concentration: 4 mM).

5 μl of a sample containing the peptide of the invention was pipettedinto a 96-well microplate. After adding 25 μl of the buffer and 10 μl ofthe enzyme, the mixture was thoroughly stirred and incubated at 37° C.for 5 minutes. After adding 10 μl of the substrate, the mixture wasreacted at 37° C. for 30 minutes. Then the reaction was ceased by adding40 μl of 0.1 N NaOH. After adding 20 μl of a methanol solution of 1%o-phthalaldehyde and allowing to stand at room temperature for 10minutes, 100 μl of 0.1 N HCl was added and the resultant mixture wasincubated at 37° C. for 30 minutes. Then the amount of His-Leu formed bythe hydrolysis by ACE was determined by exciting (at 355 nm) thefluorescent substance formed by the reaction between the amino group inthe histidine residue and o-phthalaldehyde and measuring thefluorescence wavelength at 460 nm. Then the percentage inhibition by thepeptide of the invention was determined in accordance with the followingequation and the ACE inhibitory activity (IC₅₀) was calculated.Percentage inhibition={1−(A−a)/(B−b)}×100

-   -   A: Measurement of fluorescence when the sample was added.    -   a: Measurement of fluorescence when the sample was added and the        buffer was added as a substitute for the enzyme.    -   B: Measurement of fluorescence when distilled water was added as        a substitute for the sample.    -   b: Measurement of fluorescence when distilled water was added as        a substitute for the sample and the buffer was added as a        substitute for the enzyme.

Example 1 Production and Purification of Peptide

2 L of water was added to 100 g of defatted sesame and the pH value ofthe resultant mixture was adjusted to 12.0 to 12.5 by adding NaOH. Afterstirring at 55° C. for 1 hour, the mixture was filtered to give aprotein extract. HCl was added to the protein extract to adjust the pHvalue to 4.0. After centrifuging, sesame protein (weight on dry basis:19.8 g) was obtained.

To 10 g of the obtained sesame protein, 300 ml of water was added andthe pH value of the mixture was adjusted to 7.5 with NaOH. Then 10 mg ofthermolysin (Nacalai Tesque, 7000 PU/mg) was added thereto and themixture was reacted under gentle stirring at 65° C. for 6 hours. Afterthe completion of the reaction, HCl was added to the reaction mixture toadjust to pH 4.0 and the thermolysin was inactivated by heating to 90°C. for 10 minutes. After heating, the thus formed precipitate wasremoved by centrifugation and the supernatant was filtered through paperfilter (Toyo, No. 2). The filtrate was freeze-dried to give 5.9 g of apeptide powder.

80 mg of this peptide powder was dissolved in 2 ml of 10% ethanolsolution and subjected to gel filtration column chromatography. Theconditions employed were as follows.

-   -   Column: Bio-Gel P-2 (15 mm ID×820 mm L, Bio-Rad).    -   Eluent: 10% ethanol.    -   Flow rate: 0.15 ml/min.    -   Detection: UV 210 nm.

The eluate from the column was collected in fractions at intervals of 15minutes with the use of a fraction collector. The ACE inhibitoryactivity of each fraction was measured in accordance with the methoddescribed above. As a result, the major ACE inhibitory activity underthe above conditions was observed in fractions 32 to 38. These fractionswere combined and freeze-dried. This procedure was repeated three timesand thus 37.5 mg of peptides was obtained in total.

Next, 37.5 mg of the ACE inhibitory active peptides obtained by theBio-Gel P-2 gel filtration column chromatography was dissolved in 2 mlof purified water and subjected to high-performance liquidchromatography with the use of an ODS column to thereby fractionate thepeptides. The conditions employed were as follows.

-   -   Column: Develosil ODS-10 (20 mm ID×250 mm L, Nomura Chemical).    -   Mobile phase: Buffer A: 5% CH₃CN, 0.1% TFA.        -   Buffer B: 40% CH₃CN, 0.1% TFA.    -   Gradient: 0 to 20 min: 0% Buffer B        -   20 to 80 min: 0 to 100% Buffer B.    -   Flow rate: 10 ml/min.    -   Detection: UV 210 nm.

Under the above conditions, the eluate was collected in fractions atintervals of 1 minute with the use of a fraction collector. A 5 μlportion of each fraction was pipetted into a 96-well microplate andevaporated to dryness under reduced pressure. Next, the residue wasdissolved in 5 μl of purified water to give a sample for measuring theACE inhibitory activity. Then the ACE inhibitory activity of eachfraction was measured in accordance with the method described above. Asa result, fractions 39, 52 and 54 showed strong ACE inhibitoryactivities. The 3 fractions were freeze-dried and thus a small amount ofpeptides was obtained from each fraction.

Purification of ACE Inhibitory Peptide in Fraction 39

The freeze-dried peptide of fraction 39 was dissolved in 200 μl ofpurified water and subjected to high-performance liquid chromatographywith the use of a C30 column to thereby fractionate the peptides. Theconditions employed were as follows.

-   -   Column: Develosil C30-UG-5 (10 mm ID×250 mm L, Nomura Chemical).    -   Mobile phase: Buffer: 10% CH₃CN, 0.1% TFA.    -   Flow rate: 4 ml/min.    -   Detection: UV 210 nm.

Under the above conditions, the eluate was collected in fractions atintervals of 15 seconds with the use of a fraction collector. A 5 μlportion of each fraction was pipetted into a 96-well microplate andevaporated to dryness under reduced pressure. Next, the residue wasdissolved in 5 μl of purified water to give a sample for measuring theACE inhibitory activity. Then the ACE inhibitory activity of eachfraction was measured in accordance with the method described above. Asa result, fractions 44 and 45 showed strong ACE inhibitory activities.The 2 fractions were freeze-dried separately and thus a small amount ofpeptide was obtained from each fraction. Next, these fractions weresubjected to amino acid analysis and TOF MS/MS analysis. As a result, itwas found that the peptide of fractions 44 and 45 was Leu-Ser-Ala.

Purification of ACE Inhibitory Peptide in Fraction 52

The freeze-dried peptide of fraction 52 was dissolved in 200 μl ofpurified water and subjected to high-performance liquid chromatographywith the use of a C30 column to thereby fractionate the peptides. Theconditions employed were as follows.

-   -   Column: Develosil C30-UG-5 (10 mm ID×250 mm L)    -   Mobile phase: Buffer: 14% CH₃CN, 0.1% TFA.    -   Flow rate: 4 ml/min.    -   Detection: UV 210 nm.

Under the above conditions, the eluate was collected in fractions atintervals of 15 seconds with the use of a fraction collector. A 5 μlportion of each fraction was pipetted into a 96-well microplate andevaporated to dryness under reduced pressure. Next, the residue wasdissolved in 5 μl of purified water to give a sample for measuring theACE inhibitory activity. Then the ACE inhibitory activity of eachfraction was measured in accordance with the method described above. Asa result, fractions 89 and 90 and fractions 96 and 97 showed strong ACEinhibitory activities. The 4 fractions were freeze-dried separately andthus a small amount of peptide was obtained from each fraction. Next,these fractions were subjected to amino acid analysis and TOF MS/MSanalysis. As a result, it was found that the peptide of fractions 89 and90 was Ile-Val-Tyr, while the peptide of fractions 96 and 97 wasVal-Ile-Tyr.

Purification of ACE Inhibitory Peptide in Fraction 54

The freeze-dried peptide of fraction 54 was dissolved in 200 μl ofpurified water and subjected to high-performance liquid chromatographywith the use of a C30 column to thereby fractionate the peptides. Theconditions employed were as follows.

-   -   Column: Develosil C30-UG-5 (10 mm ID×250 mm L, Nomura Chemical).    -   Mobile phase: Buffer: 17% CH₃CN, 0.1% TFA.    -   Flow rate: 4 ml/min.    -   Detection: UV 210 nm.

Under the above conditions, the eluate was collected in fractions atintervals of 15 seconds with the use of a fraction collector. A 5 μlportion of each fraction was pipetted into a 96-well microplate andevaporated to dryness under reduced pressure. Next, the residue wasdissolved in 5 μl of purified water to give a sample for measuring theACE inhibitory activity. Then the ACE inhibitory activity of eachfraction was measured in accordance with the method described above. Asa result, fractions 69 to 73 showed strong ACE inhibitory activities.The 5 fractions were separately freeze-dried and thus a small amount ofpeptide was obtained from each fraction. Next, fractions 69, 70, 72 and73 among them were subjected to amino acid analysis and TOF MS/MSanalysis. As a result, it was found that the peptide of each of thesefractions was Leu-Val-Tyr.

Example 2 Production of Peptides by Chemical Synthesis

Using an automatic peptide synthesizer (Model ABI 430) manufactured byApplied Biosystems, a purposed protected peptide resin was synthesizedby starting with the C-terminus and extending the peptide chainsuccessively by the BOC method in accordance with the program.

After the completion of the construction of the peptide on the resin,the protected peptide resin was dried. The protected peptide thusobtained was deprotected and the peptide was removed from the resinsupport by treating it with anhydrous hydrogen fluoride (HF/p-Cresol 8:2v/v, 60 minutes). The crude peptide thus obtained was extracted with 90%acetic acid and then freeze-dried to give a powdery solid. The crudepeptide thus obtained was further purified by high-performance liquidchromatography with the use of an ODS column and thus the purposedpeptide was obtained.

-   -   Column: YMC-Pack ODS-2 (30 mm ID×250 mm L, YMC).    -   Mobile phase: Buffer A: 5% CH₃CN, 0.1% TFA.        -   Buffer B: 40% CH₃CN, 0.1% TFA.    -   Gradient: 0 to 10 min: 0% Buffer B        -   10 to 90 min: 0 to 100% Buffer B.    -   Flow rate: 20 ml/min.    -   Detection: UV 220 nm.

The purity of the peptide thus purified was examined by high-performanceliquid chromatography with the use of an ODS column.

-   -   Column: Zorbax 300SB-C18 (4.6 mm ID×150 mm L, Agilent        Technologies).    -   Mobile phase: Buffer A: 1% CH₃CN, 0.1% TFA.        -   Buffer B: 60% CH₃CN, 0.1% TFA.    -   Gradient: 0 to 25 min: 0 to 100% Buffer B    -   Flow rate: 1 ml/min.    -   Detection: UV 220 nm.        Synthesis of Leu-Ser-Ala

Using Boc-Ala (BrZ) resin (0.5 mmol) as the starting amino acid resinsupport, the peptide chain was extended with the use of 2 mM portions ofamino acid derivatives Boc-Ser and Boc-Leu. Then purified Leu-Ser-Alawas obtained by the purification method described above in Example 2.The purity of the purified product measured by the method describedabove in Example 2 was 99.0%.

Synthesis of Val-Ile-Tyr

Using Boc-Tyr (BrZ) resin (0.5 mmol) as the starting amino acid resinsupport, a peptide chain was extended with the use of 2 mM portions ofamino acid derivatives Boc-Ile and Boc-Val. Then purified Val-Ile-Tyrwas obtained by the purification method described above in Example 2.The purity of the purified product measured by the method describedabove in Example 2 was 98.8%.

Synthesis of Leu-Val-Tyr

Using Boc-Tyr (BrZ) resin (0.5 mmol) as the starting amino acid resinsupport, a peptide chain was extended with the use of 2 mM portions ofamino acid derivatives Boc-Val and Boc-Leu. Then purified Leu-Val-Tyrwas obtained by the purification method described above in Example 2.The purity of the purified product measured by the method describedabove in Example 2 was 99.2%.

Example 3 Measurement of ACE Inhibitory Activity of Peptide

The ACE inhibitory activities of the 3 peptides obtained in Example 2were measured in accordance with the method described above and IC₅₀values were determined. Table 1 shows the results. As a control, the ACEinhibitory activity of the sesame peptide powder obtained in Example 1was also measured and the IC₅₀ value thereof was determined.

TABLE 1 Inhibitory activity (IC₅₀) Peptide μg/ml μM Leu-Ser-Ala 2.4 8.4Val-Ile-Tyr 1.6 4.2 Leu-Val-Tyr 0.84 2.1 Peptide powder — 50.3

Example 4 Hypotensive Effect of Peptide on Spontaneous Hypertensive Rat

SHR rats aged 17 to 22 weeks were fasted overnight. Then each of the 3peptides obtained in Example 2 was orally administered in a dose of 1mg/kg. To a control group, the same amount of water was orallyadministered for comparison. Before, and until 24 hours after theadministration, changes in systolic blood pressure were measured(BP-98A, SOFTRON). FIG. 1 shows the results.

Example 5

Using the synthetic products of Example 2, a cereal tea drink wasproduced from the following ingredients.

Composition:

roasted barley 60 g hot water 2000 ml

Peptides of Example 2

Leu-Ser-Ala 19 mg Val-Ile-Tyr 18 mg Leu-Val-Tyr 18 mgProduction Method:

Hot water was added to roasted barley and heated to 90° C. for 5minutes. After cooling to 40° C., the mixture was filtered. Then waterwas added to the extract to adjust the volume to 2000 ml. Next, theabove peptides were added and dissolved by stirring to give a cereal teadrink.

Example 6 Isolation and Quantitation of Leu-Val-Tyr from ProteinaseTreated Plant Seeds

Rice and oat grains were respectively weighed at 25 g, which were thenground to provide powders. 50 ml of hexane was added to each flower andthe solvent was removed through a filter paper (Whattman, No. 1). Thesame hexane treatment was repeated 4 times in total. Hexane was removedfrom the residue on the filter paper to provide 18.8 g of a defattedrice powder and 15.9 g of a defatted oat powder, respectively.

Each of the defatted flour was weighed at 10 g, suspended in 200 ml of0.01 N NaOH and stirred at 55° C. for 1 hour. After cooling to roomtemperature, the suspension was filtered through a filter paper(Whattman, No. 1). The filtrate was adjusted to pH 4.0 by an addition of0.1 N HCl. The precipitate thus formed was collected by centrifugation,freeze-dried to yield 0.38 g and 0.57 g of crude protein powders of riceand oat, respectively.

The obtained powder was weighed at 0.2 g, suspended in 10 ml of 0.1 mMCaCl₂. The suspension was adjusted to pH 7.5, and 0.2 mg of thermolysin(7,000 PU/mg, Nacalai Tesque) was added thereto to effect the enzymereaction under gentle mixing at 65° C. for 6 hours. After the reactionperiod, pH was adjusted to 4.0 by 1 N HCl and the thermolysin wasinactivated by heating the mixture at 90° C. for 10 minutes. Theprecipitate formed by the heating was removed by centrifugation at 3,000rpm for 30 minutes. The supernatant was freeze dried to provide peptidepowders of 28.6 mg from rice and 87.8 mg from oat.

Isolation and quantitation of Leu-Val-Tyr, as one of the peptides of theinvention, from the rice and the oat peptide powders obtained above wascarried out as follows.

i) Pre-Treatment on PD-10 Column

Each of the peptide powders of rice and oat was weighed at 20 mg,dissolved in 0.1 N acetic acid to be 5 mg/ml, filtered through amicro-filter (Millex-HV, pore size 0.45 μm, filter diameter 13 mm,Millipore Corporation) to remove insoluble components. A 2.5 ml portionof the filtrate was introduced into a PD-10 column (desalting column,Amersham Biosciences) equilibrated with 0.1 N acetic acid. The columnwas washed with a further 3.5 ml volume of 0.1 N acetic acid. Then thefraction eluted with an additional 3.0 ml volume of 0.1 N acetic acidwas collected, evaporated to dryness, dissolved in 0.5 ml of water andthen freeze-dried.

ii) Gel Filtration HPLC with the Use of TSK-GEL G2000SWXL

The specimen prepared by the pre-treatment on the PD-10 column wasdissolved in 250 μl of 45% CH₃CN, 0.1% TFA to be centrifuged at 2,000rpm for 5 minutes. The filtrate was filtered through a micro-filter(Millex-HV, pore size 0.45 μm, filter diameter 13 mm, MilliporeCorporation) to remove insoluble components.

A 50 μl portion of the filtrate was charged to a column of TSK-GELG2000SWXL (7.8×300 mm, Tosoh Corporation) equilibrated with 45% CH₃CN,0.1% TFA, and HPLC was performed with 45% CH₃CN, 0.1% TFA (flow rate 0.7ml/min., detection wavelength 280 nm). The eluate of 1 minute between 30seconds before and after the retention time was collected, evaporated todryness, dissolved in 0.5 ml of water and freeze-dried. The retentiontime of Leu-Val-Tyr was pre-determined by separately subjectingsynthetic Leu-Val-Tyr to HPLC under the same conditions.

iii) Reverse HPLC on Develosil C30-UG-5 (Quantitation of Leu-Val-Tyr)

Leu-Val-Tyr in the active peptide fractions from the gel filtration wasquantitatively analyzed by reverse HPLC on a Develosil C30-UG-5 column(3×150 mm, Nomura Chemical Co., Ltd.). The fraction from the gelfiltration HPLC on TSK-GEL G2000SWXL was dissolved in 250 μl of 5%CH₃CN, 0.1% TFA, centrifuged at 2,000 rpm for 5 minutes, and thesupernatant was filtered through a micro-filter (Millex-HV, pore size0.45 μm, filter diameter 13 mm, Millipore Corporation) to removeinsoluble components. A 50 μl portion of the filtrate was charged on aDevelosil C30-UG-5 column equilibrated with 5% CH₃CN, 0.1% TFA toperform a chromatography under the following conditions:

Elution Solvent

-   -   0-5 min.: 5% CH₃CN, 0.1% TFA    -   5-10 min.: 5-14% CH₃CN, 0.1% TFA    -   10-35 min.: 14% CH₃CN, 0.1% TFA

Flow rate: 0.4 ml

Detection wavelength: 280 nm

The peak of the each peptide of rice and oat in the Develosil C30-UG-5column chromatography, of which the retention time corresponded to thatof the authentic Leu-Val-Tyr of the peptide, was collected. The fractionwas subjected to a TOF MS analysis and a TOF MS/MS analysis to confirmthat the fraction was Leu-Val-Tyr.

A calibration curve was prepared by charging different amounts of theauthentic Leu-Val-Tyr to the same Develosil C30-UG-5 column under thesame conditions as described above and plotting the peak areas againstthe charged amounts.Calibration Curve Y=249197X−2150.6(R2=0.9991)

-   -   Y: peak area, X: amount of Leu-Val-Tyr (μg)

The peak areas of the Leu-Val-Tyr fractions from the Develosil C30-UG-5chromatography of rice and oat were applied to the calibration curve. Asa result, the amounts of Leu-Val-Tyr in 1 mg of the peptide from riceand oat were determined to be 0.71 μg and 1.05 μg, respectively.

1. An edible composition containing a purified tripeptide consisting ofan amino acid sequence of Leu-Val-Tyr.
 2. An edible compositionaccording to claim 1, comprising from 0.001 mg to 100 mg of saidtripeptide in a single intake dose.
 3. An angiotensin converting enzymeinhibitor comprising from 0.001 mg to 100 mg of a purified tripeptideconsisting of an amino acid sequence of Leu-Val-Tyr in a single dose fororal administration.
 4. A hypotensive agent comprising from 0.001 mg to100 mg of a purified tripeptide consisting of an amino acid sequence ofLeu-Val-Tyr in a single dose for oral administration.
 5. A method fortreating hypertension, wherein the method comprises administering theedible composition of claim
 1. 6. A method for inhibiting an angiotensinconverting enzyme, wherein the method comprises allowing a purifiedtripeptide consisting of an amino acid sequence of Leu-Val-Tyr to act onthe enzyme.
 7. A method for suppressing elevation of blood pressure,wherein the method comprises administering the edible composition ofclaim 1.